1. Technical Field
The present disclosure relates to methods and products that facilitate simultaneous detection of multiple genetic alterations in multiple different genes.
2. Background Information
Genetic mutations play a key role in the pathogenesis, progression and drug resistance of cancer and various infectious diseases. Although some genes have a predilection for involvement in certain cancers and drug-induced mutations, there is significant variation among patients, and multiple different genes could be involved. This underscores the need for a simple method that facilitates simultaneous examination of multiple genes. However, such a goal is very difficult to accomplish due to the complexity of the human genome and the presence of sequence homologues. In order to assure the specificity of mutation detection and enhance sensitivity, current assays require sequence-specific amplification for each target of interest. Likewise, mutations conferring drug resistant microorganisms vary among patients, and various microorganism subtypes can have similar clinical presentations. As a result, screening for mutations in infectious microorganisms also requires laborious multiple amplifications and sequencing. Moreover, the sensitivity of most assays is limited to approximately 1-10%. Therefore, the presence of mutants with a low incidence cannot be detected unless a more sensitive assay becomes available.
The present disclosure describes a universalized strategy for simultaneous screening of a multitude of multiple different genetic regions of interest through the use of two ubiquitous adapters designated as a “blocking adapter” and a “reporter adapter,” respectively. In normal wild-types, the blocking adapter is the sole adapter being ligated. In the presence of mutants, sequential ligation of the blocking adapter and the reporter adapter is enabled by differential sequence fill-in (DSF) with a complementary deoxyribonucleotide triphosphate (dNTP), followed by protection from single-stranded DNA-specific nuclease digestion and full-length sequence extension. This leads to the displacement of downstream RNA and the sense strand of the blocking adapter, allowing the creation of a 3′ overhang for ligation with the reporter adapter to form mutant-dual adapter hybrids. Mutants and homologous sequences that are not completely filled in with complementary dNTPs are digested into fragments by single-strand-specific nuclease, thereby preventing them from undergoing full-length sequence extension and ligation with the reporter adapter. By targeting the reporter adapter or mutant-dual adapter hybrids, mutants filled in with a complementary dNTP are detected/quantified or amplified for sensitive detection of low frequency mutants. Of note is that all different mutant-dual adapter hybrids share the same sequence at the dual adapter ligation site regardless of the origin or the number of mutations involved. Therefore, mutation detection may be easily augmented by polymerase chain reaction (PCR) with the use of just two ubiquitous primers: one derived from the blocking adapter and the other from the reporter adapter. Likewise, other sequence amplification methods, such as ligase chain reaction (LCR) or transcription-mediated amplification (TMA), may also be directed to target the commonly shared sequence. Without the need of multiple target-specific sequence amplifications, the methods described in this disclosure make it a simple task to perform high throughput mutation screening for a multitude of multiple different genes in a multitude of multiple different samples.